5th Bilateral Symposiumwfischer/conference/2011-05-Bilateral...Dr. Stefanie Eschenlohr, DAAD Information Centre Taipei, Director Prof. Dr. Wolfgang Fischer, National Yang-Ming University,

NYMU - HD 2013

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5th Bilateral Symposium

National Yang-Ming University and Heidelberg University

5. – 6. October 2012

National Yang-Ming University, Taipei, Taiwan

NYMU - HD 2013

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NYMU - HD 2013

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Sponsors:National Yang-Ming University

National Science Council Taiwan

Biophysical Society of R.O.C.

Molecular Biophysics

Spectroscopy TechniqueDrug Development

Nano Medicine

Sessions

For registration email: [email protected]

http://www.ym.edu.tw/~wfischer/international_vmp_conf/

Design by M

eng-Han Lin

National Yang-Ming University, Taipei, Taiwan

SpeakersShy Arkin , Jerusalem, IL Arthur Chiou , TaipeiChristoph Cremer , Mainz, DERainer Fink , Heidelberg, DEWolfgang Fischer , TaipeiEric Freed , NIH/NCI, Frederick, MD, USAStephen Hashmi , Heidelberg, DE Dieter Heermann , Heidelberg, DEAndreas Herrmann , Berlin, DEYi-Tsau Huang , Taipei

Hidekatsu Iha , Oita, JPFu-Jen Kao , Taipei Bernd König, Jülich, DEJung-Hsin Lin , Taipei Alex Ma , Taipei Klaus Strebel , NIH, Bethesda, USABing Sun ,Pasteur Institute, ShanghaiPeter Tieleman , Calgary, CA Chin-Tien Wang , Taipei

Plenary LectureHarald zur Hausen

Nobel Laureate in Medicine 2008

Viral Membrane Proteins - Taipei 2012

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Symposium on Viral Membrane Proteins

National Yang-Ming University

5. – 6. October 2012

Taipei, Taiwan


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Schedule: Thursday, 4th of October 2012 Morning Arrival Evening 18:00 – 18:30 Registration DAAD, Taipei 101 18:30 – 18:50 Welcome and Introduction Dr. Michael Zickerick, German Institute Taipei, Director General Dr. Stefanie Eschenlohr, DAAD Information Centre Taipei, Director Prof. Dr. Wolfgang Fischer, National Yang-Ming University, Taipei 18:50 – 19:45 Lecture Prof. Harald zur Hausen, DKFZ, Heidelberg, DE Cancer prevention by vaccination. 19:45 German Buffet Dinner Friday, 5th of October 2012 8:15 Hotel Shuttle Bus to National Yang-Ming University (NYMU) 8:30 – 9:00 Registration Opening 9:00 – 9:10 President Liang, National Yang-Ming University Director General Dr. Michael Zickerick, German Institute Taipei 9: 10 – 10:00 Prof. Harald zur Hausen, DKFZ, DE, Nobel Laureate 2008

The search for infectious causes of human cancers. 10:00 – 10:25 Alex Ma, Taipei

The future Influenza vaccine: monoglycosylated hemagglutinin. 10:25 – 10:50 Shy Arkin, Jerusalem, IL

How do aminoadamantanes block the influenza M2 channel and how does resistance develop?


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10:50 – 11:00 Coffee Break 11:00 – 11:25 Peter Tieleman, Calgary, CN Simulation studies on the role of the M2 proteins in viral budding.

11:25 – 11:50 Andreas Herrmann, Berlin, DE Influenza virus binds to its host cell through multiple dynamic interactions – A single virus force spectroscopy and force probe MD simulation study.

11:50 – 12:15 Klaus Strebel, Bethesda, USA

Functional Characterization of HIV-1 Vpu. 12:15 – 12:40 Wolfgang Fischer, Taipei Multi scale simulations on the assembly of viral channel forming proteins. 12:40 – 14:00 Lunch Break 14:00 – 14:25 Eric Freed, Frederick, USA

HIV-1 assembly and release.

14:25 – 14:50 Bernd König, Jülich, D Interaction of HIV-1 VpU and human CD4.

14:50 – 15:15 Bing Sun, Shanghai.

ORF4a protein of HCoV-229E forms an ion channel functionally analogous to the SARS-CoV 3a protein.

15:15 – 15:40 Chin-Tien Wang, Taipei Identifying SARS-CoV membrane protein amino acid residues linked to virus-like particle assembly.

15:40 – 16:00 Coffe Break 16:00 – 16:25 Dieter Heermann, Heidelberg, DE A topological similarity measure for proteins. 16:25 – 16:50 Jung-Hsin Lin, Taipei

An anatomy of membrane protein dynamics.

16:50 – 17:15 Yi-Tsau Huang, Taipei

Bioactivity study of herbs against hepatic fibrogenesis induced by hepatocyte apoptotic bodies.

18:00 – 19:30 Dinner at Escargot, NYMU


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Saturday, 6th of October 2012 8:15 Hotel Shuttle Bus to NYMU 9:00 – 9:10 Opening 9:10 – 9:35 Stephen Hashmi, Heidelberg, DE

The organic side of gold, palladium and platinum. 9:35 – 10:00 Rainer Fink, Heidelberg, DE The ryanodin-receptor - a potential target for VMPs? 10:00 – 10:25 Fu-Jen Kao, Taipei

Long working distance fluorescence lifetime imaging with stimulated emission and electronic time delay.

10:25 – 10:45 Coffee Break 10:45 – 11:10 Christoph Cremer, Mainz, DE

Far field fluorescence microscopy of viral proteins at molecular optical resolution.

11:10 – 11:35 Hidekatsu Iha, Oita, JP

Application of the novel lectin microarray system for the diagnosis of ATL pathogenesis.

11:35 – 12:00 Arthur Chiou, Taipei

Optical tweezers based bio-micro-rheology.

12:00 – 12:15 Closing Remarks 12:15 – 14:00 Lunch Break Afternoon Tour: National Palace Museum, Taipei Evening Dinner


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Participants: Keynote Speaker Prof. Dr. Harald zur Hausen Deutsches Krebsforschungszentrum Im Neuenheimer Feld 280 DO 02.049 69120 Heidelberg, Germany T. +49 6221 42 4804 F. +49 6221 42 4808 E-Mail: [email protected] Speakers Prof. Dr. Isaiah Arkin Vice President Institute of Life Sciences Department of Biological Chemistry The Hebrew University, Givat-Ram Jerusalem, 91904, Israel Tel./Fax: +972 – (0)2 – 6584329 Email: [email protected] Prof. Dr. Arthur Chiou Dean of International Affairs, NYMU Director, Biophotonics and Medical Imaging Research Center Institute of Biophotoncis School of Biomedical Science and Engineering National Yang-Ming University 155, Sec. 2, Li-Nong St. Taipei 112, Taiwan Tel.: +886 2 2826 Email: [email protected]


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Prof. Dr. Christoph Cremer Institute of Molecular Biology gGmbH (IMB) Ackermannweg 4 55128 Mainz, Germany Applied Optics & Information Processing, Kirchhoff Institute for Physics, and Institute for Pharmacy and Molecular Biotechnology Heidelberg University Im Neuenheimer Feld 227 D-69120 Heidelberg , Germany Tel.: +49 6221 549252 (Secretary: -54-9271) Fax: +49 6221 549112 Email: [email protected] Prof. Dr. Rainer Fink Medicinal Biophysics Institute for Physiology und Pathophysiology Heidelberg University Im Neuenheimer Feld 326 D-69120 Heidelberg, Germany Tel.: +49 6221 54 4065 or 4084 Fax: +49 6221 54 4123 Email: [email protected] Prof. Dr. Wolfgang B. Fischer Institute of Biophotonics School of Biomedical Science and Engineering National Yang-Ming University 155, Sec. 2, Li-Nong St. Taipei 112 Tel.: +886 2 2826 7394 Fax: +886 2 2823 5460 Email: [email protected] Prof. Dr. Eric O. Freed Head, Virus-Cell Interaction Section HIV Drug Resistance Program National Cancer Institute Frederick National Laboratory for Cancer Research P.O. Box B, Building 535, Room 110 Frederick, MD 21702-1201, USA Tel.: +1 301-846-6223 (office); +1 301-846-6483 (lab) Fax: +1 301-846-6777 E-mail: [email protected]


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Prof. Dr. A. Stephen K. Hashmi Dean of Faculty of Chemistry and Geo-Sciences Organisch-Chemisches Institut Heidelberg University Im Neuenheimer Feld 270 D-69120 Heidelberg, Germany Tel.: +49(0)6221-54 8413 Fax: +49(0)6221-54 4205 Email: [email protected] Prof. Dr. Dieter W. Heermann Institut für Theoretische Physik Philosophenweg 19 Heidelberg University D-69120 Heidelberg, Germany Tel.: +49 6221 549 431 Fax: +49 6221 549 331 Email: [email protected] Prof. Dr. Andreas Herrmann Department of Biology, Lab Molecular Biophysics Humboldt-University Berlin, Invalidenstr. 42, 10115 Berlin, Germany Tel.: +49 030-2093-8830 Fax: +49 030-2093-8585 Email: [email protected] Prof. Dr. Yi-Tsau Huang Director National Research Institute of Chinese Medicine 155, Sec. 2, Li-Nong St. Taipei 112, Taiwan Tel.: +886 2 2820 3101 Fax: +886 2 2823 30488 Email: [email protected] Prof. Dr. Hidekatsu Iha Department of Microbiology Oita University Faculty of Medicine 1-1 Idaiga-oka, Hasama Yufu 879-5593, Oita, Japan. Tel.: +81-97-586-6224 Fax: +81-97-586-5719 Email: [email protected]


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Prof. Dr. Fu-Jen Kao Institute of Biophotonics School of Biomedical Science and Engineering 155, Sec. 2, Li-Nong St. Taipei 112, Taiwan Tel.: +886 2 2826 7394 Fax: +886 2 2823 5460 Email: [email protected] Dr. Bernd W. König Institute of Complex Systems: Strukturbiochemie (ICS-6) Wilhelm-Johnen-Straße 52425 Jülich, Germany Telefon: +49 2461 61-5385 Fax: +49 2461 61-8766 E-Mail: [email protected] Prof. Dr. Jung-Hsin Lin Research Center for Applied Sciences Academia Sinica 128 Sec. 2, Academia Rd., Nankang, Taipei 11529 Tel: + 886-2-27898000 ext 63 Email: [email protected] Prof. Dr. Alex Che Ma Genomics Research Center Academia Sinica 128 Academia Road, Section 2, Nankang, Taipei, 115, Taiwan Tel.: +886-2-27871233 Email: [email protected] Prof. Dr. Bing Sun Co-Director Laboratory of Molecular Virology Chinese Academy of Science Institute Pasteur of Shanghai 225 South Chongqing Road Shanghai 200025 Tel.: +86 (0)21-63851927 Fax: +86 (0) 21-63843571 Email: [email protected]


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Prof . Dr. Klaus Strebel Chief, Viral Biochemistry Section Laboratory of Molecular Microbiology National Institute of Allergy and Infectious Diseases National Institute of Health Building 4 Center Drive MSC Room 312 0460, Bethesda, MD 20892-0460 Tel.: +1 301-496-3132 Fax: +1 301-402-0226 Email: [email protected] Prof. Dr. Peter Tieleman Dept. of Biological Sciences, BIOL 415 University of Calgary 2500 University Dr. NW Calgary, Alberta Canada, T2N 1N4 Tel: +1 (403) 220-2966 Fax: +1 (403) 289-9311 Email: [email protected] Prof. Dr. Chin-Tien Wang Institute of Clinical Medicine National Yang Ming University, and Department of Medical Research and Education Taipei Veterans General Hospital 201, Sec, 2, Shih-Pai Road, Beitou Taipei 112, Taiwan Tel.: +886-2-28712121 ext.2684 Fax: +886-2-28742279 Email: [email protected]


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Abstracts:


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How do aminoadamantanes block the influenza M2 channel and how does resistance develop?

Isaiah Arkin

Institute of Life Sciences Department of Biological Chemistry The Hebrew University, Givat-Ram

Jerusalem, 91904, Israel Email: [email protected]

The interactions between channels and their cognate blockers are at the heart of numerous biomedical phenomena. Herein, we unravel one particularly important example bearing direct pharmaceutical relevance: the blockage mechanism of the influenza M2 channel by the anti-flu amino-adamantyls (amantadine and rimantadine) and how the channel, and consequently the virus develop resistance against them. Using both computational analyses and experimental verification, we find that amino-adamantyls inhibit M2's H+ channel activity by electrostatic hindrance due to their positively charged amino group. In contrast, the hydrophobic adamantyl moiety on its own, does not impact conductivity. Additionally, we were able to uncover how mutations in M2 are capable of retaining drug binding on the one hand, yet render the protein, and the mutated virus, resistant to amino-adamantyls, on the other hand. We show, that the mutated, drug resistant protein has a larger binding pocket for the drug. Hence, despite binding the channel, the drug remains sufficiently mobile so as not to exert a H+-blocking positive electrostatic hindrance. Such insight into the blocking mechanism of amino-adamantyls, and resistance thereof, may aid in the design of next generation anti-flu agents.


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Optical Tweezers Based Bio-Micro-rheology.

Arthur Chiou

Institute of Biophotonics

Biophotonics & Molecular Imaging Research Center National Yang-Ming University, Taipei, Taiwan

Email [email protected]

Bio-Micro-Rheology (BMR) refers to the study of the viscoelastic properties of biological samples, such as bio-fluids, cells, nucleic acids and proteins, at the micron or sub-micron scale. Viscoelastic properties of a myriad of biological cells and bio-fluids are strongly correlated to their physiological functions; a change in their viscoelastic properties, even at a very small fraction on the order of a few percents, is often concomitant with related diseases. A classical example is the red blood cells (RBCs) whose deformability is critical to their oxgen-carrying and delivery function through veins and arteries. In the case of biological fluids, the viscosity of blood is closely related to cerebral vascular disease and coronary artery disease, and the liquefaction of vitreous humor can lead to retinal detachment. The relation of viscoelastic alterations of cerebral-spinal fluid and hydrocephalus has also been reported. Accurate measurement of the viscoelastic properties of biological samples at either single cell resolution (for the case of biological cells) or with sample volume on the order of micro-liter (for the case of biological fluids) may hence shed light on clinically-relevant mechano-biology at the molecular level. From the practical point of view, the requirement of only a very small amount of sample is particularly important since many biological fluids such as synovial fluid and vitreous humor are available only in very limited amount.

Optical-Tweezers Based Micro-Rheology has emerged in recent years as one of the critical techniques capable of fulfilling the needs elucidated above. In this talk I will introduce 4 complementary approaches for bio-microrheology, namely (1) dynamic light scattering (or diffuse wave spectroscopy); (2) blinking optical tweezers based single-particle tracking; (3) oscillatory optical tweezers based biomicrorheology; (4) jumping optical tweezers based biomicrorheology. Applications of these techniques to a wide range of samples including individual human red blood cells; macrophage cells, DNA, synovial fluid, and polymer solutions such as polysodium styrene sulfonate (NaPSS) and hyaluronic acids will be highlighted in this talk. Keywords: Bio-microrheology, optical tweezers, viscoelasticity


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Farfield Fluorescence Microscopy of viral Proteins at molecular optical Resolution.

Christoph Cremer

Lightoptical Nanoscopy, Institute of Molecular Biology (IMB), D-55128 Mainz/Germany; Institute for Pharmacy and Molecular Biotechnology (IPMB)/Kirchhoff-Institute of Physics,

University Heidelberg, D-69120 Heidelberg/Germany Email: [email protected]

Novel developments in optical technology and photophysics made possible to radically overcome the resolution limit (ca. 200 nm laterally, 600 nm along the optical axis) of conventional far field fluorescence microscopy1. Here, we report on the use of a particular mode of Spectrally Assigned Localization Microscopy (SALM), Spectral Precision Distance/Position Determination Microscopy (SPDM)2,to analyze virus related protein distributions at the single molecule resolution level. SPDM permits the application of conventional fluorophores together with standard preparation conditions, thus highly simplifying the analysis3. - For example, SPDM allowed the quantitative analysis of the spatial arrangement of individual YFP (yellow fluorescent protein) tagged gp36.5/m164 proteins derived from murine cytomegalovirus proteins in COS-7 cells (collaboration with R. Holtappels, Institute of Virology, University Mainz, Germany, and M. Hausmann/P. Mueller, University Heidelberg, Germany3). In other applications, we examined the feasibility to use SPDM for the nanoscopic analysis of the interaction of influenza viruses with the membrane of human epithelial lung cells (collaboration with C. Ehrhardt/R. Dierkes, Institute of Molecular Virology, University Muenster, Germany). The present results indicate that SPDM may be favourably applied to study with single molecule resolution the spatial distribution of influenza viral proteins at the cell membrane, as well as the spatial distribution of potential receptor clusters on the membrane. In the latter case, the SPDM method was applied to study the distribution of individual fluorescence labelled HGFR proteins in PR8 virus infected cells (Q. Wang et al., ms in preparation). - In addition to the study of viral protein expression and the interaction of viruses and membranes, SPDM may be applied even to the nanostructural analysis of individual viruses (collaboration with C. Wege, Plant Virology, University Stuttgart). As a well known model system, Tobacco mosaic virus (TMV) particles were chosen. SPDM of directly fluorescence labeled TMV coat proteins allowed (with an error in the 3 nm range) to measure correctly the diameter (18 nm) of the TMV viruses (M. Gunkel et al., ms in preparation). 1C. Cremer, Optics far Beyond the Diffraction Limit: From Focused Nanoscopy to Spectrally

Assigned Localization Microscopy (2012). In: Springer Handbook of Lasers and Optics, 2nd edition (F. Träger, Edit.), pp. 1351 – 1389. 2P.Lemmer, M.Gunkel, D.Baddeley, R. Kaufmann, A. Urich, Y. Weiland, J.Reymann, P. Müller,

M. Hausmann, C. Cremer (2008) SPDM – Light Microscopy with Single Molecule Resolution at the Nanoscale. Applied Physics B 93: 1-12. 3C. Cremer, R. Kaufmann, M. Gunkel, S. Pres, Y. Weiland, P. Müller, T. Ruckelshausen, P.

Lemmer, F. Geiger, S. Degenhard, C. Wege, N.-A. W. Lemmermann, R. Holtappels, H. Strickfaden, M. Hausmann (2011) Superresolution imaging of biological nanostructures by spectral precision distance microscopy, Biotechnology 6: 1037–1051.


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Electromechanical coupling in muscle: a viral target?

Rainer Fink

Medicinal Biophysics Institute for Physiology und Pathophysiology

University of Heidelberg Im Neuenheimer Feld 326

D-69120 Heidelberg, Germany Email: [email protected]

Contraction of striated as well as smooth muscle cells is regulated by changes in the myoplasmic free calcium concentration. The initial step of the excitation-contraction coupling involves different cellular mechanisms to achieve the necessary calcium concentration changes. A depolarisation of the membrane potential of the muscle cells can -depending on the type of muscle- either directly stimulate calcium inflow into the myoplasm and/or lead to a release of calcium ions from intracellular stores, in particular the sarcoplasmic or endoplasmic reticulum. Furthermore, it is now widely accepted that calcium regulation can be profoundly disturbed by viral attacks of cells. However, there is still very little known about possible viral-induced changes in excitation-coupling, in particular of cardiac and skeletal muscle.


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Multi scale simulations on the assembly of viral channel forming proteins.

Wolfgang B. Fischer

Institute of Biophotonics

School of Biomedical Science and Engineering National Yang-Ming University, Taipei, Taiwan.

E-mail: [email protected] Enveloped viruses encode a series of membrane proteins which allow their survival within the cell. Amongst these proteins are those which are known to form channels or pores. In recent years evidence has mounted that some of these proteins not only form channels but also interact with host factors, which are also membrane proteins. Questions arise whether the functional form generating channels is the same as when interacting with the host factors? A series of computational tools are used to address this question using Vpu from HIV-1 as an example. Vpu is an 81 amino acid bitopic type I integral membrane protein of HIV-1. It is responsible for the enhancement of viral release by interacting with host factors, eg. CD4, Bst-2 and NTB-A, and forming channels. Combining docking approaches, classical and coarse grained molecular dynamics simulations the route of a transmembrane domain of Vpu mimicking its biophysical journey through the cell and its interactions, with itself, ions and host factors, is outlined.


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HIV-1 Assembly and Release.

Eric O. Freed

Virus-Cell Interaction Section, HIV Drug Resistance Program,

National Cancer Institute, Frederick, MD, 21702 Email: [email protected]

The HIV-1 Gag polyprotein precursor directs the assembly and release of virus particles from the infected cell surface. The N-terminal matrix (MA) domain of Gag is the primary determinant of Gag-membrane localization. Although it is clear that, in most instances, HIV-1 assembly takes place predominantly on the plasma membrane (PM), Gag’s trafficking itinerary remains incompletely understood. We previously reported that HIV-1 assembly takes place in cholesterol-enriched PM microdomains (rafts) and that the phospholipid PI(4,5)P2 is an important cellular cofactor in directing Gag to the inner leaflet of the lipid bilayer. We are currently investigating the role that several cellular protein families play in Gag trafficking; these include the ADP ribosylation factors (ARFs), the SNAREs, and F-BAR domain-containing proteins. We find that siRNA-mediated depletion, or overexpression of WT, dominant-active, or dominant-negatives versions of these proteins, disrupts Gag-membrane binding. In addition to directing the Gag precursor to the PM, the MA domain also plays a key role in the virion incorporation of the viral envelope (Env) glycoprotein complex, which is composed of the surface glycoprotein gp120 and the transmembrane glycoprotein gp41. In the past, we have described a number of single amino acid mutations in the MA domain that block Env incorporation. These genetic data support the existence of a direct interaction between MA and the cytoplasmic tail of gp41. However, we have now identified an amino acid substitution in MA that globally rescues the Env incorporation defect imposed by the above-mentioned MA mutations. Recent findings regarding host cell machinery involved in Gag trafficking, and the molecular mechanism of HIV-1 Env glycoprotein incorporation into virus particles, will be highlighted in my talk.


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The Organic Side of Gold, Palladium and Platinum.

A. Stephen K. Hashmi

Organisch-Chemisches Institut Ruprecht-Karls-Universität Heidelberg

Im Neuenheimer Feld 270 D-69120 Heidelberg, Germany

Email: [email protected]

The influence of organometallic chemistry in different sectors will be presented. a) Gold as a catalyst allows the preparation of pharmaceutically relevant structures by reaction pathways, which seem to contradict classical text book knowledge. This has made gold a major source of innovation in the catalysis research of the last decade. b) Another part of the presentation will cover new types of organopalladium and organoplatinum compounds, which can be generated with the help of strained organic molecules. The interactions of these new types or organometallic compounds with biological systems were be investigated.


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A Topological Similarity Measure for Proteins.

Andreas Hofmann, Nicolas Wenzel, Gabriell Mate, Dieter W. Heermann

Institut für Theoretische Physik Philosophenweg 19

Heidelberg University D-69120 Heidelberg, Germany

Email: [email protected] Motivated by fact that proteins responsible for organizing the folding and hence the 3D structure of chromosomes are very flexible we have developed a similarity measure for proteins and ligands based on concepts from topology. This measure allows measuring the physical similarity on the basis of topological properties, i.e., neighborhood properties rather than on euclidean properties. Hence the measure can deal with uncertain atomic or larger molecular complex positions as well as for example flexible parts of proteins and ligands. We show that the measure is well defined and gives results compatible with other measures on a test suit derived from the database DUD (A Directory of Useful Decoys).


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Influenza virus binds to its host cell through multiple dynamic interactions – A single virus force spectroscopy and force probe MD Simulation study.

Andreas Herrmann

Humboldt-University Berlin, Department of Biology, Lab Molecular Biophysics

Invalidenstr. 42, 10115 Berlin, Germany Email: [email protected]

Influenza virus belongs to a wide range of enveloped viruses. Virus-host cell binding marks the first critical step of infection. Hence, forces involved in this process are essential. The major spike protein hemagglutinin binds sialic acid residues of glycoproteins and -lipids with dissociation constants in the millimolar range (Sauter et al. (1992) Biochemistry 31:9609–9621), indicating a multivalent binding mode. We characterized the attachment of influenza virus to host cell receptors using three independent approaches. Optical tweezers and atomic force microscopy based single molecule force spectroscopy provides powerful tools to measure binding forces in biological systems. Optical tweezers and atomic force microscopy based single molecule force spectroscopy revealed very low interaction forces. The observation of sequential unbinding events strongly suggests a multivalent binding mode between virus and cell membrane. However, an assignment of forces to their underlying molecular interactions involved in these processes is difficult or even cannot be obtained by these techniques. In molecular dynamics, time-dependent interactions between all atoms within a given system are calculated numerically. Force probe molecular dynamics simulations extends this method by introducing a moving harmonic potential as a "virtual spring" acting on given atoms Molecular dynamics simulations reveal a variety of unbinding pathways that indicate a highly dynamic interaction between HA and its receptor allowing to rationalize influenza virus-cell binding quantitatively at molecular level.


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Bioactivity study of herbs against hepatic fibrogenesis induced by hepatocyte apoptotic bodies.

Ting-Fang Lee1, Yun-Lian Lin2, and Yi-Tsau Huang1,2*

1Institute of Traditional Medicine, National Yang-Ming University, Taipei, Taiwan 2National Research Institute of Chinese Medicine, Taipei, Taiwan


Hepatic stellate cells (HSCs), the key cell type for hepatic fibrosis, become activated and profibrogenic in the presence of hepatocyte apoptotic bodies (ABs). Bupleurum scorzonerifolium (BS), a widely used traditional Chinese herb for liver diseases, was fractionated, and the inhibitory effects of BS extracts on AB-induced HSC migration were screened. The activity-guided fractionation led to a lignan, kaerophyllin. The current study evaluated the in vivo role of kaerophyllin in protecting the liver against injury and fibrogenesis caused by thioacetamide (TAA) in rats and further explored the underlying mechanisms. Sprague-Dawley rats liver fibrosis was induced by intraperitoneal injection of TAA twice per week for 6 weeks. Kaerophyllin (10 or 30 mg/kg) was given by gavage twice per day consecutively for 4 weeks starting 2 weeks after TAA injection. Rat HSCs were used to investigate the anti-inflammatory role of kaerophyllin against TNF-α in vitro. PPAR-γ expression was knocked down in rat HSCs using PPAR-γ small interfering RNAs. Kaerophyllin significantly protected liver from injury by reducing serum AST, ALT levels, and by improving the histological architecture and fibrosis score. In addition, kaerophyllin suppressed inflammation by reducing the mRNA of TNF-α, IL-1β and MCP-1 genes. In HSCs, kaerophyllin elevated PPAR-γ activity and reduced TNF-α-stimulated mRNA levels of ICAM-1, MCP-1 and IL-1β genes, which were reversed by siRNA knock-down of PPAR-γ gene. Our results demonstrated that kaerophyllin protected the rat liver from TAA-caused injury and fibrogenesis by suppressing hepatic inflammation and inhibiting HSC activation, possibly through upregulation of PPAR-γ expression.


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Application of the novel lectin microarray system for the diagnosis of ATL pathogenesis.

Hidekatsu Iha

Department of Microbiology, Oita University Faculty of Medicine, Idaigaoka, Hasama, Yufu 879-5593, JAPAN.


Regardless of species, all the living organisms have poly-saccharides called glycans in their cellular surfaces or even inside the cells. Glycans interact with sugar-binding proteins, lectin, and affects on the numerous biological activities such as development, immune response, organogenesis or even cancer development. Although there are many reports on the expressional disorders of lectins or glycans in cancer cells, most of them have described only single or few gene product's aberrations. Here we describe the application of a novel lectin microarray system (LecMS), which consists of 45 lectins with different binding preferences and provides the multiple glycan-lectin binding intensity information, on various leukemic or lymphoma cell lines. As we carry the endemic areas of Adult T-cell Leukemia (ATL), which is caused by an infection of retrovirus (HTLV-1), our interest was focused on how this system characterize the ATL or non-ATL cells by their glycan-lectin binding intensity values. From the results of LecMS profiling on thirteen different ATL or non-ATL tumor cells, we found lectin values which are commonly induced in all tumor cells and values induced specifically in ATL cells, respectively. High-throughput, sensitive and relatively simple nature of this system can be utilized as the novel diagnostic tool for the prognosis of ATL pathogenesis.


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Long working distance fluorescence lifetime imaging with stimulated emission and electronic time delay.

Po-Yen Lin1,2, Shin-Shian Lee1, Chia-Seng Chang2, and Fu-Jen Kao1*

1Institute of Biophotinics, Nation Yang-Ming University, Taipei 11221, Taiwan, ROC 2Institute of Physics, Academia Sinica, Taipei 11529, Taiwan, ROC

*Email: [email protected]

In this work, long working distance fluorescence lifetime imaging is realized with stimulated emission in combination with electronic time delay control. Spatial coherence, as a result of stimulated emission, enables unattenuated fluorescence detection at extended distance, using low NA optics. The electronic time delayed trigger provides an advantageous way in adjusting the pulse separation and probing the fluorescence lifetime in the nanosecond ranges. The lifetime of selected fluorophores is accurately determined through the pump-probe configuration. The characteristics and applications in lifetime measurement of stimulated emission are investigated and summarized succinctly here.


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Interaction of HIV-1 VpU and human CD4.

Bernd W. König

Institute of Complex Systems: Strukturbiochemie (ICS-6) Wilhelm-Johnen-Straße 52425 Jülich, Germany


Viral protein U (VpU) of human immunodeficiency virus type 1 (HIV-1) plays an important role in downregulation of the main HIV-1 receptor CD4 from the surface of infected cells. Physical binding of VpU to newly synthesized CD4 in the endoplasmic reticulum is an early step in a pathway leading to proteasomal degradation of CD4. We identified regions in the cytoplasmic domain of VpU that are involved in CD4 binding by NMR spectroscopy. Amino acids in both helices found in the cytoplasmic region of VpU in membrane mimicking detergent micelles experience chemical shift perturbations upon binding to CD4, while amino acids between the two helices and at the C-terminus of VpU show no or only small changes, respectively. The topology of the complex was further studied using paramagnetic relaxation enhancement. Paramagnetic spin labels were attached at three sequence positions of a CD4 peptide comprising the transmembrane and cytosolic domains of the receptor. VpU binds to a membrane proximal region in the cytoplasmic domain of CD4.


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An Anatomy of Membrane Protein Dynamics.

Jung-Hsin Lin1,2,3*, Eric Minwe Liu1, Yu-Hsuan Chen3

1Division of Mechanics, Research Center for Applied Sciences, Academia Sinica; 2Institute of Biomedical Sciences, Academia Sinica; 3School of Pharmacy, National Taiwan University;

E-mail address: [email protected]

Dynamics is a crucial part for understanding the function of a biomolecule. Although information about intramoelcular dynamics is difficult to obtain with experiments, it is rather accessible via molecular dynamics simulations. Recently, microsecond time scale for simulations of membrane proteins, namely, receptors, transporters, pumps, have become widely affordable, and it is therefore timely to ask whether molecular dynamics simulations at this time scale can already provide sufficient dynamical information ready for functional interpretation. Standard analyses for dynamics of biomolecules include atomic fluctuations, dynamic cross-correlation matrix (DCCM), principal component analysis, etc. However, with the substantial lengthening of time scale, these analyses should be performed with care, especially when molecule conformations are represented in cartesian coordinates. In this talk, we will demonstrate how more appropriate analyses could be performed with suitable structural alignments, guided by statistical tests. Besides, with the time-lag dynamical cross correlation matrix (tlDCCM) analysis, we will show how long the correlation can persist. We also will illustrate how a membrane protein can be anatomized in terms of dynamics of correlated motions. Specific examples include the human adenosine A2A receptor bound with different ligands, the bacterial efflux pump Sav1866 transporter, and the bacterial leucine transporter LeuT.


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The future Influenza vaccine: monoglycosylated hemagglutinin.

Alex Che Ma

Genomics Research Center Academia Sinica, Taipei, TW


The outbreak of 2009swine-origin influenza A (H1N1) virus has caused a global pandemic during whichincreased numbers of patients were diagnosed with influenza-like illness anddeaths. Influenza pandemics occur when Hemagglutinins (HA) are little or notrecognized by immunity and the viruses transmit efficiently from human tohuman. HA is the major viral surface glycoprotein that binds to specificsialylated glycan receptors in the respiratory tract and allows the virus toenter the cell. It has been recognized as the key antigens in the host immune responseto influenza virus in both natural infection and vaccination. We aim to investigate the effect of HAglycosylation on immune response and to develop a cross-protective vaccine. Theanalysis of HA sequences revealed that the peptide sequences around theglycosylation sites are highly conserved, and a single N-linked GlcNAc at each glycosylation sites on HA(monoglycosylated HA, HAmg) as a protein vaccine increased the antibodies response andneutralization activity against influenza subtypes than the fully glycosylatedHA (HAfg). Inthis study, we examined the cross reactive and protective anti-HA antibodyelicited by vaccination of HA, with or without glycans, adjuvanted with alumtoward 2009 swine-origin influenza A (H1N1) virus. The results showed that theHAmg vaccine elicited anti-HA antibodies with higherhemagglutination inhibition, microneutralization activity and better survivalrate in virus challenge experiments than HAfg. The protein vaccinesof HAmg designed from 2009 A (H1N1) swine and 2007 A (H1N1) Brisbaneare both sufficient to provide cross-protection against infections with thehighly pathogenic H1N1 viruses. This study provides new insights into the crossreactive and protective immunity provided by HAmg and opens a newstrategy for influenza vaccine design.


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Functional Characterization of HIV-1 Vpu.

Klaus Strebel

Laboratory of Molecular Microbiology, NIAID, NIH; Bethesda, MD (USA) Email: [email protected]

HIV-1 Vpu is an 81 amino acid type 1 integral membrane protein that has been shown to cause proteasomal degradation of CD4 but also enhances the release of virions from infected cells. These two biological activities of Vpu are mechanistically distinct and involve different structural domains in Vpu. Vpu does not affect assembly of viral particles but facilitates the detachment of virions from the cell surface. Recently, BST-2 (also known as tetherin, CD317, or HM1.24 in the literature) was identified as the host factor responsible for the Vpu-sensitive restriction of HIV-1 virus release. BST-2 does not selectively inhibit the release of HIV-1 virions but affects the release of a number of enveloped viruses, such as PERV, Ebola, Lassa, Marburg, endogenous betaretrovirus of sheep (enJSRV), or KSHV. We have performed numerous biochemical and virological studies to understand the mechanism of BST-2 mediated inhibition of virus release and to gain insights into how Vpu interferes with this function of BST-2. I will summarize our current understanding of the functional interaction of Vpu and BST-2 and discuss structural features in BST-2 required for virus tethering.


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ORF4a protein of HCoV-229E forms an ion channel functionally analogous to the SARS-CoV 3a protein.

Bing Sun

Laboratory of Molecular Virology

Institute Pasteur of Shanghai Chinese Academy of Science

Shanghai 200025 Email: [email protected]

Previously, we found that there is a specific gene in all coronavirus genomes termed the SNE gene. SNE protein from SARS-CoV, which is also called orf3a, forms an ion channel and regulates the SARS-CoV release. Here we study the ORF4a of Human Coronavirus 229E (HCoV-229E) which encodes a homologues protein of SRAS-CoV 3a. Computer programs predict that ORF4a protein consisting of four TM domains, homodimers and tetramers can be detected by co-immunoprecipitation. Two-electrode voltage clamp (TEVC) experiment demonstrated that SNE protein of HCoV-229E forms a monovalent cation channel in Xenopus laevis oocytes, which is permeable to potassium and sodium ions. Furthermore, after HUH-7 cells were transfected with siRNA, which is known to suppress ORF4a expression, followed by infection with HCoV-229E, the released virus was significantly decreased. This finding will help to understand the role of ion channel in coronavirus life cycle. Keywords: human coronavirus 229E; ORF4a; ion channel Reference: [1] Zeng R, Yang RF, Shi MD et al. 2004. Characterization of the 3a protein of SARS-associated coronavirus in infected vero E6 cells and SARS patients. J Mol Biol. 341(1):271-9. [2] Lu W, Zheng BJ, Xu K et al. 2006. Severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release. Proc Natl Acad Sci U S A. 103(33):12540-5. [3] Wang K, Lu W, Chen J et al. 2012. PEDV ORF3 encodes an ion channel protein and regulates virus production. FEBS Lett. 586(4):384-91.


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Simulation studies on the role of the M2 protein in viral budding.

Eduardo Mendez-Villuendas and Peter Tieleman

Dept. of Biological Sciences, BIOL 415 University of Calgary, Alberta, Canada

Email: [email protected] The budding of enveloped viruses is a complex multi-step process requiring alterations in membrane curvature and scission at the neck of the budding virion. M2 is a pH-dependent matrix protein from influenza virus widely known for its role in viral uncoating and the target of the amantadine flu drug that prevents proton transport. An additional role played by M2 relies on collective effects where M2 clusters have been hypothesized to induce local membrane curvature, resulting in a reduced energetic cost associated with the bending of the membrane and where the budding of virus particles takes place in a cholesterol dependent manner (Rossman et al., Cell 142, pp. 902-913, 2010). We are using computer simulations to study the effect of M2 tetramers on lipid mixtures consisting of sphingomyelin, DOPC and cholesterol in different ratios and temperatures in a range where lipid mixing and segregation take place. We are working on estimating entropic and free energy contributions to membrane curving and other energetic and structural effects through large-scale simulations with the coarse-grained MARTINI model. These models will form the basis for understanding in detail how M2 induces curvature in membranes as part of the viral budding mechanism in influenza.


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Identifying SARS-CoV Membrane Protein Amino Acid Residues Linked to Virus-Like Particle Assembly.

Chin-Tien Wang

Institute of Clinical Medicine, National Yang Ming University, and Department of Medical Research and Education Taipei Veterans General Hospital

Taipei 112, Taiwan

Severe acute respiratory syndrome coronavirus (SARS-CoV) membrane (M) proteins are capable of self-assembly and release in the form of membrane-enveloped vesicles and of forming virus-like particles (VLPs) when coexpressed with SARS-CoV nucleocapsid (N) protein. According to past deletion analyses, M self-assembly involves multiple M sequence regions. To identify important M amino acid residues for VLP assembly, we coexpressed N with multiple M mutants containing substitution mutations at the amino-terminal ectodomain, carboxyl-terminal endodomain, or transmembrane segments. Our results indicate that a dileucine motif in the endodomain tail is required for efficient N packaging into VLPs. Some substitution mutations led to significantly reduced VLP yields—largely due to defective M secretion. Our results suggest that aromatic residue retention at specific positions is critical to M function in terms of directing virus assembly.


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